Healthy Prenatal Dietary Pattern and Offspring Autism

Key Points Question What is the association between a healthy prenatal dietary pattern and offspring autism diagnosis and autism-associated traits in 2 large prospective cohort studies: the Norwegian Mother, Father, and Child Cohort (MoBa) and the Avon Study of Parents and Children (ALSPAC)? Findings In this cohort study including 84 548 mother-infant dyads in MoBa and 11 670 mother-infant dyads in ALSPAC, maternal consumption of a healthy dietary pattern was associated with reduced likelihood of offspring autism diagnosis (MoBa) and reduced likelihood of social communication difficulties (MoBa and ALSPAC). No other consistent associations were observed. Meaning These findings highlight the association between prenatal diet and offspring autism-related outcomes and contribute to the evolving understanding of autism etiology.


Introduction
The prevalence of autism spectrum disorder diagnosis is estimated to be 1% to 2% in the general population. 1,2In this study, we use autism, in response to the preferences of the autistic community. 3tism diagnosis reflects a heterogeneous spectrum of neurodevelopmental conditions characterized by persistent difficulties and differences in reciprocal social communication and restricted and repetitive behaviors and interests. 4These autism-associated traits extend to subclinical manifestations commonly referred to as the broader autism phenotype 5 and are key areas of development with relevance across the broader population. 6Furthermore, the relationship between social communication difficulties and restrictive and repetitive behaviors are phenotypically and genetically dissociable. 7Therefore, it may be advantageous to etiological understanding to measure autism diagnosis and autism-associated traits in the population, including the subdomains of social communication difficulties and restrictive and repetitive behaviors.
Prenatal dietary patterns are an emerging plausible exposure in the complex etiology of autism, yet this evidence base is limited.Previous studies have largely focused on discrete facets of prenatal nutrition and found autism diagnosis and autism-associated traits were inversely associated with prenatal multivitamin and/or folic acid supplement use, adequate vitamin D status, and high prenatal fish intakes. 8Yet, nutrients have synergistic and antagonistic effects, the summation of which can be measured through prenatal dietary patterns, which could broaden our etiological perspective and complement investigations of discrete nutrients and prenatal multinutrient supplements. 9 our knowledge, only 4 studies have investigated the associations of prenatal dietary patterns with autism diagnosis or autism-associated traits.However, the sample sizes were small [10][11][12][13] and results may have been affected by recall bias. 12,13Small sample sizes can increase the risk of type I and type II errors and inflated effect estimates.Furthermore, imprecise measures of diet and autismassociated traits increase random error, which requires larger sample sizes to detect an association, should one exist.Thus, while each study had strengths, their limitations may create heterogeneous results.Therefore, we sought to build on this evidence and measured the associations of high adherence to a healthy prenatal dietary pattern, compared with low adherence, with autism diagnosis and autism-associated traits in 2 large prospective cohort studies, the Norwegian Mother,

Study Population
The study is reported following the Strengthening the Reporting of Observational Studies in Epidemiology-Nutritional Epidemiology (STROBE-nut) extension of the STROBE statement.We separately analyzed ALSPAC and MoBa to test the consistency of results across contexts, although to ease comparability, we harmonized the analytical approach used in each cohort, where possible.

MoBa is a population-based pregnancy cohort study conducted by the Norwegian Institute of Public
Health. 14 ALSPAC is a prospective cohort primarily of pregnant women and their offspring.Pregnant individuals resident in the Southwest of England with expected dates of delivery between April 1,  1991, and December 31, 1992.Of 20 248 eligible pregnancies, 14 541 pregnant individuals (71.8%) participated in ALSPAC.Further details have been published elsewhere. 15,16Details of all the data are available through a fully searchable data dictionary. 17Ethical approval for the study was obtained from the ALSPAC Ethics and Law Committee and the local research ethics committees.Informed consent for the use of data collected via questionnaires was implied by return of a completed postal questionnaire following the recommendations of the ALSPAC Ethics and Law Committee at the time.
For our use of MoBa and ALSPAC data, we restricted to plausible food frequency questionnaire (FFQ) responses and singleton pregnancies, which left 84 548 pregnancies in MoBa and 11 760 pregnancies in ALSPAC (Figure 1).In MoBa, FFQ responses were only available for recruitment years 2002 to 2008. Figure 2 shows the source and timeline of all data collection.

Outcomes Autism Diagnosis
The Norwegian Patient Registry was linked to all MoBa children via unique identification number, capturing all diagnoses of autism made in the public specialist health care system from 2008 to 2018.
Children were up to age 16 years at diagnosis.Autism diagnosis is recorded using the International Statistical Classification of Diseases and Related Health Problems, Tenth Revision (ICD-10) criteria, and we included all F84 codes except Rett syndrome (F84.2).

Autism-Associated Traits
From MoBa, we used the 40-item Social Communication Questionnaire (SCQ) about autismassociated social communication difficulties (SCQ-SOC) and restrictive and repetitive behaviors (SCQ-RRB). 18The SCQ was completed by the mother about their child at 2 time points, ages 3 and 8 years.Item 1 of the questionnaire screens for phrase speech and is not scored, and we restricted to those with more than 50% response rate. 19We applied an adjustment for absence of phrase-speech as per Eaves et al, 20 as children without phrase-speech may be underscored due to omission of inapplicable items.As secondary outcomes, we analyzed the social communication and restrictive and repetitive behaviors subdomains.From ALSPAC we used the 12-item Social and Communication Disorders Checklist (SCDC), which was completed by the primary caregiver about their child at approximately age 7.6 years.The SCDC is a questionnaire that measures difficulties with social and communication skills.High scores indicated greater autism-associated traits and were defined as at least 93rd percentile for SCDC at age approximately 8 years, SCQ at age 3 years, and SCQ at age 8 years and was guided by previous validation studies for MoBa 19 and ALSPAC. 21

Dietary Assessment and Dietary Pattern Analyses
Both MoBa and ALSPAC used a self-reported FFQ for which detailed methods have been previously published.3][24][25] ALSPAC applied a nonquantitative FFQ that asked pregnant participants to report their current food intake in relation to 43 food groups. 26,27MoBa and ALSPAC collected information on commonly consumed foods and beverages and dietary supplement use.Food intake was expressed as frequency of consumption in ALSPAC and grams per day in MoBa.
We matched the food and beverage items used in ALSPAC and MoBa and derived a healthy dietary pattern from each cohort using exploratory factor analysis with varimax rotation (eAppendix 1, eTable 1, and eTable 2 in Supplement 1).The derived factors were selected based on interpretability and Scree plot with eigen values greater than 1.5.The linear factor score was adjusted for total energy intake using the residuals method 28 and categorized into terciles as high, medium, and low adherence to a healthy prenatal dietary pattern (HPDP).The pattern was denoted healthy as food items with high factor scores represent foods that are encouraged according to dietary guidelines worldwide and nationally. 29,30The dietary patterns generally loaded highly for fruits, vegetables, fish, nuts, and whole grain foods and had low factor loadings for red and processed meats, soft drinks, and foods high in fats and/or refined carbohydrates.Additionally, nutrient intakes across the levels of adherence to HPDP (eTable 3 in Supplement 1) were broadly consistent with each country's respective national nutritional recommendations.See eAppendix 1 in Supplement 1 for further details.

Statistical Analysis
The analytic strategy was planned a priori and conducted separately in ALSPAC and MoBa; however, the same analytic approach was applied.Descriptive statistics were presented for both the cohorts overall and by tercile of HPDP.Potential confounders were identified with a directed acyclic graph 31,32 (eFigure 1 and eAppendix 2 in Supplement 1) and existing literature. 1 Covariates were maternal age, maternal education, maternal depression, prepregnancy body mass index (BMI; calculated as weight in kilograms divided by height in meters squared), planned pregnancy, prenatal multivitamin use, prenatal alcohol intake, prenatal smoking, prepregnancy hypertension, and prepregnancy diabetes (Table 1; eAppendix 3 in Supplement 1).The minimally sufficient adjustment set was adjusted for using inverse probability weights estimates through entropy balancing. 33Weights and covariate balance were checked (<0.1 negligible covariate imbalance). 34Within a generalized nonlinear model, we further clustered on the mother to account for the intercorrelation between siblings.Robust standard errors were estimated using Horvitz-Thompson variance estimator to account for the inverse probability weights and clustering. 35,36All statistical models were 2-tailed, with α = .05.We used R Studio version 4.0.38][39] Data analysis occurred February 1, 2022, to August 1, 2023.
Data were assumed to be missing at random and imputed using multivariate imputation by chained equations. 38We included all variables in the models and a range of auxiliary variables (eTable 4 in Supplement 1).We used 115 imputations for ALSPAC and 156 imputations for MoBa, selected based on the von Hippel approach, 40 and imputed each level of HPDP separately to facilitate the testing of interaction terms. 41The weights and marginal structural models were estimated for each imputed dataset 42 before combining them using Rubin rule to produce the single and final estimate of association. 43The characteristics of participants with missing outcome information are presented in eTables 5 to 7 in Supplement 1.
In sensitivity analyses, we repeated the main analyses making changes to the confounding, outcome, and exposure measurements (eAppendix 4 in Supplement 1).For confounding, we adjusted for additional covariates, including paternal characteristics and birth year, and assessed the estimates without adjustment for prenatal multivitamin supplement use.For sensitivity analysis of outcomes, we applied an alternative approach to score the SCQ and described the overlap in children with high scores for SCQ at ages 3 and 8 years and autism diagnosis.For sensitivity analysis of our exposure, we replaced the HPDP with each of its subgroups, plant-based, fish-based, and unhealthy dietary patterns (eTable 2 in Supplement 1).Interactions tested were between HPDP and prepregnancy BMI (<25 vs Ն25), child's sex (male or female), multivitamin supplement use (yes or no) and maternal education (MoBa: less than college or university degree or a degree or higher; ALSPAC: less than A-level or greater than A-level).Lastly, complete-case analyses were conducted.The sociodemographic characteristics varied across cohorts and within cohorts (Table 1; eTable 8 in Supplement 1).Compared with ALSPAC participants, MoBa participants were older at recruitment, had higher educational levels, and were more likely to have planned their pregnancy, abstain from alcohol, and use a multivitamin supplement.MoBa and ALSPAC participants with high HPDP adherence, compared with those with low adherence, were more likely to be older, with high educational attainment, use prenatal multivitamin supplements, and be nonsmokers.In ALSPAC only, high HPDP adherence was associated with greater prevalence of alcohol consumption in pregnancy and lower prevalence of a planned pregnancy and history of depression.Good covariate balance was achieved, and there were no extreme weights (eFigure 2 and eFigure 3 in Supplement 1).

MoBa
The event rate for each outcome (autism diagnosis from SCQ at ages 3 and 8 years or SCDC at age 8 years) was only higher in the lowest level of adherence to a HPDP compared with medium or high adherence (Table 2).The proportions of children with recurrent high scores across the outcomes were modest but was especially low for SCQ-RRB at age 3 years (eTable 9 in Supplement 1).

Discussion
Our cohort study found a lower likelihood of autism diagnosis and autism-associated traits for offspring of mothers with high adherence to HPDP, albeit inconsistently for measures of autismassociated traits.This inconsistency related to differential associations with the subdomains and at different ages.Overall, more consistent results were observed for social communication difficulties.
Lastly, there was weak evidence of association modification by child sex, but inconsistent evidence with the other characteristics, maternal education, prepregnancy BMI, and prenatal supplement use.
Our large prospective investigation makes an important contribution to existing literature.
Previous studies have largely focused on multinutrient supplements, discrete nutrients, or food groups. 8Yet, diet makes the largest contribution to overall nutrient intakes and captures the totality of complex nutrient interactions. 9Moreover, there is a small body of existing literature on prenatal diet and autism-associated outcomes, and results are conflicting.We addressed some limitations of previous investigations, such as small sample size and retrospective study design, so that our analyses may provide more reliable results.
Two retrospective studies found a healthy prenatal diet was associated with a lower likelihood of autism diagnosis, 12,13 but a prospective investigation did not replicate their findings. 10However, the retrospective investigations had a high risk of bias, especially recall bias, 12,13 and most studies included fewer than 100 children with autism diagnosis. 10,12We only identified 2 previous prospective investigations into autism-associated traits (measured linearly), but their results were conflicting.The first study investigated 2 cohorts with 727 and 154 mother-child dyads and measured offspring social responsiveness scores between ages 4 to 18 years and 36 months, respectively. 10Of the 6 prenatal dietary patterns investigated, none were clearly associated with autism-associated traits in the final adjusted model. 10The second prospective study, including 325 mother-child dyads, observed less autism-associated traits at age 12 to 14 months if mothers had a greater Mediterranean dietary pattern score.Autism-associated traits were measured using an adapted version of the Infant Toddler Social and Emotional Assessment questions. 11We obtained more precise estimates, yet our main results on autism-associated traits were also conflicting.
We considered whether the subdomains or child's age at measurement were potential sources of heterogeneity.The variable findings on autism-associated traits may relate to the performance of the SCQ at ages 3 or 8 years and SCDC at age 8 years.In addition to autism diagnosis, the SCQ and SCDC detect children with greater social communication difficulties or inflexible behaviors and interests that do not necessarily constitute a diagnosable condition. 19,21This is particularly true in children younger than 4 years, especially for restrictive and repetitive behaviors, which are a common feature of typical development. 44We estimated that only approximately one-fifth of children with a high SCQ score at age 3 years continued to score highly at age 8 years, although this estimate may be underestimated due to missing data at age 8 years.A validation study in MoBa confirmed the SCQ-RRB did not discriminate between autistic and nonautistic children at age 3 years, whereas the SCQ-SOC performed fairly well. 19Additionally, we compared the full SCQ (39 items) and SCDC, as they are designed to screen for autism, 19,21 yet the SCDC only measures social communication skills. 21Overall, social communication difficulties were more consistently associated with HPDP.
We observed the association of HPDP with social communication skills in both cohorts, despite variability in the composition of HPDP between MoBa and ALSPAC.6][47] Similar consistency in diet-outcome associations across dietary pattern measures are observed in other fields. 45,48It has been suggested that this may relate to broadly similar core components of a healthy diet, such as high intakes of fruits, vegetables, and whole grains and low intakes of animal products and highly processed foods. 49 observed inconsistent evidence that female offspring, compared with male offspring, had a greater reduction in the likelihood of social communication difficulties at age approximately 8 years if their mothers had consumed an HPDP.This was replicated across MoBa and ALSPAC but only in relation to the highest adherence to HPDP and not at age 3 years.Despite considerable interest in a theorized female protective effect, few investigations test for association modification by child sex. 50[56] The male preponderance of autism diagnosis contributes to debates of a female protective effect.Several mechanisms have been proposed and relate to an extreme male brain, sex hormones, genetics, and immune function. 50However, misclassification bias and sex differences in developmental trajectories may impact results.The conceptualization of autism has been predominantly based on White males, which has led to underdiagnosis in females and can impact the performance of autism-screening tools. 57Additionally, many features of communication develop earlier in girls compared with boys, 57 which may further explain our conflicting results between ages 3 and 8 years.Overall, social communication skills may be more strongly associated with HPDP in females, but the mechanisms remain to be determined.
Lastly, although nutrients are distributed across multiple foods, there are key food sources of specific nutrients.In MoBa, and to a lesser extent in ALSPAC, plant-based foods, compared with fishbased and unhealthy foods, had clearer associations with social communication difficulties.There are several explanations for the variation across food groups, such as differences in random error and bias structures, larger number of food items, or a true effect of plant-based foods.We identified a previous study that directly compared food groups while simultaneously adjusting for other foods; however, the results were inconclusive, possibly due to limited statistical power to detect an association. 58Further studies are required to clarify if plant-based foods have clearer associations with social communication difficulties.
Whether the associations observed are causal remains to be established.The etiology of autism has been linked to several pathways, such as genetics, maternal immune activation, sex hormones, the microbiome, and environmental factors. 51It is hypothesized that prenatal diet may alter DNA methylation patterns, 11 regulate immune processes, or interact with toxins. 8

Strengths and Limitations
Our study had many strengths, such as the large prospective design, cross context comparison, analyses of subdomains of autism-associated traits, dietary subgroups, and interactions.We carefully considered potential confounders and adjusted for a wide range, including paternal characteristics, and further tested this through sensitivity analysis.However, unmeasured, and residual confounding can still occur, for example, genetic confounding, parental caregiving or childhood diet.A further strength of our investigation was the analysis of detailed dietary information collected prospectively during pregnancy.3][24][25] We adjusted for a proportion of error through the residuals method. 28ditionally, a single assessment of maternal diet is a limitation and precludes the evaluation of dietary changes over time.Although some dietary changes during pregnancy may occur, misclassification would likely bias results toward the null.
Even autism diagnosis is broadly defined, and diagnostic criteria and practices have changed over time. 58

Conclusions
In this cohort study of mother-child dyads, the likelihood of autism diagnosis was reduced by 22% in association with high adherence to HPDP.Regarding autism-associated traits, we observed an association between social communication difficulties and higher HPDP adherence but inconsistent associations with restrictive and repetitive behaviors.

Father
, and Child Cohort Study (MoBa), and the Avon Longitudinal Study of Parents and Children (ALSPAC).
Participants were recruited from all over Norway from 1999 to 2008.Of 277 702 eligible pregnancies, 95 200 pregnant individuals (41%) consented to participation.The cohort includes approximately 114 500 children, 95 200 mothers, and 75 200 fathers.Our study is based on version 12 of the quality-assured data files released for research in January 2019.The establishment of MoBa and initial data collection were based on a license from the Norwegian Data Protection Agency and approval from the Regional Committees for Medical and Health Research Ethics.All participants provided written informed consent.The MoBa cohort is currently regulated by the Norwegian Health Registry Act.Our use of MoBa data was approved by the Regional Committees for Medical and Health Research Ethics.

Figure 1 .
Figure 1.Flowchart of Sample Selection in Norwegian Mother, Father, and Child Cohort (MoBa) and Avon Longitudinal Study of Parents and Children (ALSPAC)

Figure 2 .
Figure 2. Timeline and Source of Data Collection in Norwegian Mother, Father, and Child Cohort (MoBa) and Avon Longitudinal Study of Parents and Children (ALSPAC)

Table 1 .
Sociodemographic Characteristics by Adherence to a Healthy Prenatal Dietary Pattern in MoBa and ALSPAC JAMA Network Open.2024;7(7):e2422815. doi:10.1001/jamanetworkopen.2024.22815(Reprinted) July 18, 2024 6/15 Downloaded from jamanetwork.comby guest on 07/25/2024 There were lower odds of each outcome in the crude models in association with HPDP (autism diagnosis: OR, 0.68 [95% CI, 0.61-0.83];SCQ at age 3 years: OR, 0.86 [95% CI, 0.79-0.93];SCQ at age 8 years: OR, 0.80 [95% CI, 0.72-0.88];SCDC at age 8 years: OR, 0.59 [95% CI, 0.48-0.74])(eTable 10 in Supplement 1).Higher HPDP adherence was associated with a lower likelihood of autism diagnosis in MoBa and lower SCDC score at age 8 years in ALSPAC, yet no clear evidence of association was observed for SCQ at ages 3 or 8 years in MoBa (Figure 3).In MoBa, higher HPDP adherence was associated with a lower likelihood of a high score on SCQ-SOC at age 3 years but higher likelihood of a high score on SCQ-RRB at age 3 years.Results were robust to adjustment for additional covariates (eTable 11 in Supplement 1).Further sensitivity analysis indicated that, generally, the clearest associations in HPDP were in analyses of the food subgroups.The likelihood of a high score on SCQ at age 8 years (OR, 0.92 [95% CI, 0.86-0.99]),and SCQ-SOC at age 8 years (OR, 0.89 [95% CI, 0.82-0.96])reduced in association with a plant-based dietary pattern, and the likelihood of a high score on SCQ-RRB at age 8 years (OR, 0.93 [95% CI, 0.88-0.99])reduced in association with a fish-based dietary pattern.Compared with the HPDP analysis, the differences were small (eTable 12 in Supplement 1).Evidence for interaction was unclear and/or inconsistent across MoBa and ALSPAC, except between HPDP and child sex (SCQ-SOC at age 3 years, SCQ-SOC at age 8 years, and SCDC at age 8 years) (eTable 13 in Supplement 1).A greater magnitude of association was observed in female offspring in relation to SCQ-SOC at age 8 years and SCDC at age 8 years and for male offspring in relation to SCQ-SOC at age 3 years.The complete-case analyses in MoBa produced similar results to the main analyses (eTable 14 in Supplement 1).

Table 1 .
Sociodemographic Characteristics by Adherence to a Healthy Prenatal Dietary Pattern in MoBa and ALSPAC (continued) Prepregnancy BMI was modeled linearly but BMI categories are presented descriptively to aid interpretation.

Table 2 .
Autism Diagnosis and Autism-Related Traits by Adherence to a Healthy Prenatal Dietary Pattern in ALSPAC and MoBa a The presence of the outcome from each questionnaire was indicated by a high score.

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14,15able pregnancies, systematic differences in recruitment and retention, especially in the autismassociated traits analysis due to the high attrition rate.14,15Motherswith low adherence to HPDP and offspring with high SCQ scores at age 3 years had a greater proportion of missing data for SCQ at age 8 years.This further indicates selection bias, which would bias toward the null and reduce generalizability.
These factors increase heterogeneity, reduce precision, and increase bias, which may have affected our results.Selection bias may affect our findings through the inevitable exclusion of JAMA JAMA Network Open.2024;7(7):e2422815.doi:10.1001/jamanetworkopen.2024.22815(Reprinted) July 18, 2024 10/15 Downloaded from jamanetwork.comby guest on 07/25/2024 Furthermore, we observed that female offspring may have a greater magnitude of association of adherence to HPDP with social communication difficulties at age 8 years.At present, we remain uncertain as to whether the associations observed are causal.Further research should substantiate our findings, especially given the inconsistency in the previous literature and across our measures of autism-associated traits.It would be advantageous to measure the subdomains combined and separately and explore whether associations differ by food group.Additionally, triangulation with alternative study designs and exploration of potential mediators is required to support causal interpretation of the associations observed in our study.Food Items and Factor Loadings Representing a Healthy Prenatal Dietary Pattern eTable 2. Food Items and Factor Loadings of the Subgroups of a Healthy Prenatal Dietary Pattern eTable 3.Estimated Nutrient Intakes in ALSPAC and MoBa by Level of Adherence to a Healthy Prenatal Dietary Pattern eAppendix 2. Explanation of Directed Acyclic Graph eFigure 1. Directed Acyclic Graph of Relationship Between Healthy Prenatal Dietary Pattern and Autism-Related Outcomes eAppendix 3. Covariates eAppendix 4. Sensitivity Analyses eTable 4. Variables Used in Imputation Models eTable 5. Baseline Characteristics of Pregnancies With Missing Outcome Data at Age Three in MoBa eTable 6. Baseline Characteristics of Pregnancies With Missing Outcome Data at Age Eight in MoBa eTable 7. Baseline Characteristics of Pregnancies With Missing Outcome Data at Age Eight in ALSPAC eTable 8. Descriptive Statistics for Additional Sociodemographic Characteristics Used in Sensitivity Analyses eTable 9. Number and Proportion of Children Across Each Outcome in MoBa eFigure 2. Covariate Balance Plot for Adherence to a Healthy Prenatal Dietary Pattern in ALSPAC eFigure 3. Covariate Balance Plot for Adherence to a Healthy Prenatal Dietary Pattern in MoBa eTable 10.Unadjusted Measures of Association Between a Healthy Prenatal Dietary Pattern and Autism Diagnosis and Autism-Associated Traits eTable 11.Associations Between a Healthy Prenatal Dietary Pattern and Autism Diagnosis and Autism-Associated Traits in MoBa and ALSPAC With a Different Adjustment Set of Covariates eTable 12. Associations Between a Healthy Prenatal Dietary Pattern and Food Groups With Autism Diagnosis and Autism-Associated Traits in ALSPAC and MoBa eTable 13.Associations Between a Healthy Prenatal Dietary Pattern With Autism Diagnosis and Autism-Associated Traits in MoBa and ALSPAC Stratified by Prenatal Multivitamin Supplement Use, Prepregnancy BMI, Child Sex, and Maternal Education eTable 14.Complete Case Analyses Measuring the Associations Between Adherence to a Healthy Prenatal Dietary Pattern and Autism Diagnosis and Autism-Associated Traits in MoBa eReferences.